The Ca +/calmodulin-dependent protein kinase II (CaMKII) is an important mediator of cellular Ca + signals. CaMKII is required for forms of learning and memory and is involved in regulation of dendritic morphology, synapse number and synaptic strength. CaMKII has also been implicated in cell cycle control, insulin secretion, and in ischemia-induced neuronal cell death. Many studies of CaMKII have relied on the pharmacological inhibitor KN93. However, KN93 also inhibits CaMKFV and, more importantly, voltage-dependent Ca2+ channels. Peptide inhibitors of CaMKII related to AIP are widely believed to be more specific, however, they also inhibit other CaM kinases as well as PKA. Thus, a better inhibitor would be a valuable and widely applicable tool. Such an inhibitor should be (i) CaMKII-specific, (ii) cellpermeable to allow easy use in cellular assays, (iii) readily available at reasonable cost or effort, and (iv) should be well characterized to allow educated interpretation of obtained data. As a starting point for such an inhibitor, a naturally occurring CaMKII inhibitory protein termed CaM-KIIN is a very promising source, as it does not affect activity of CaMKIV, PKC or PKA. In this proposal, we will first use peptides derived from the CaM-KIIN sequence to determine the minimally required region necessary for efficient and specific inhibition of CaMKII. We will then make cell permeable peptides based on such minimal inhibitory region, using the permeabilizing ant and tat sequences. The efficiency and specificity of these peptides will first be determined in biochemical assays. We will then determine their effect on substrate phosphorylation within neurons and a non-neuronal expression system; a suitable substrate to be tested is the AMPA-type glutamate receptor subunit GluR1. Then, the inhibitor will be tested for its action on the different modes of CaMKII activity (calcium/calmodulin-stimulated, autonomous by autophosphorylation, and autonomous by NR2B binding). The inhibitor developed and characterized in this proposal will allow easy testing of specific CaMKII functions in various physiological and pathological situations, and may thus lead to new therapeutic avenues, including for stroke.